Mitochondrial dysfunction is a common contributing factor to degenerative diseases, including the neural and muscular systems; yet mechanistic studies in mammals are limited because adequate tools and approaches are lacking. Moreover, a greater understanding of the protein translocation mechanisms in model organisms such as S. cerevisiae is needed, because protein translocation is linked to metabolism, signaling, and mitochondrial quality control and stress pathways such as mitophagy. The overall goals of this proposal are (1) to increase our mechanistic understanding of protein import pathways into mitochondria in both yeast and mammalian cells and (2) to understand how defects in protein translocation contribute to disease. Attenuating protein translocation pathways can alter the location of mitochondrial proteins that are dual-localized (located to mitochondria and another compartment), change mitochondrial stress pathways, and induce selective turnover of mitochondria via mitophagy. We have a large collection of small molecules that we have identified from various chemical genetic screens that can be used to modulate protein translocation pathways in yeast, cultured cells, and animal models. To accomplish our proposal goals, we have identified three specific study aims. In Aim 1, we will characterize how small molecule modulators for the TIM22 import pathway impair protein import and characterize the assembly of a novel mitochondrial carrier that is linked to a new mitochondrial disease. In Aim 2, small molecules from a new chemical genetic screen for the TOM/TIM23 import pathway will be characterized in mechanistic studies and in disease studies, particularly focusing on how defects in protein import alter mitochondrial stress pathways. In addition to increasing our fundamental knowledge about the mechanisms of protein translocation into mitochondria, this application may have a broad impact on public health because our approach will provide new tools that will provide a strategy for developing therapeutics for diseases that can be modified by attenuating protein translocation.